Apparatus and method for separating and concentrating fluids containing multiple components

ABSTRACT

An apparatus is disclosed that allows for separating and collecting a fraction of a sample. The apparatus, when used with a centrifuge, allows for the creation of at least three fractions in the apparatus. It also provides for a new method of extracting the buffy coat phase from a whole blood sample. A buoy system that may include a first buoy portion and a second buoy member operably interconnected may be used to form at least three fractions from a sample during a substantially single centrifugation process. Therefore, the separation of various fractions may be substantially quick and efficient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/932,882, filed Sep. 2, 2004, now U.S. Pat. No. 7,374,678,issued on May 20, 2008, entitled “APPARATUS AND METHOD FOR SEPARATINGAND CONCENTRATING FLUIDS CONTAINING MULTIPLE COMPONENTS”, which is acontinuation-in-part of U.S. patent application Ser. No. 10/445,381,filed May 23, 2003, now U.S. Pat. No. 7,179,391, issued on Feb. 20,2007, entitled “APPARATUS AND METHOD FOR SEPARATING AND CONCENTRATINGFLUIDS CONTAINING MULTIPLE COMPONENTS” that claimed the benefit of U.S.Provisional Application No. 60/383,013, filed on May 24, 2002. Thedisclosures of the above applications are incorporated herein byreference.

FIELD

The present invention relates to a multiple component fluid and aconcentrator/separator, and more particularly relates to a containeroperable with a centrifuge to separate and concentrate variousbiological components.

BACKGROUND

Various fluids, such as whole blood or various other biological fluidsmay be separated into their constituent parts, also referred to asfractions or phases. For example, whole blood samples may include aplurality of constituents that may be separated by density in a devicesuch as a centrifuge. The whole blood sample may be placed in a testtube, or other similar device, which is then spun in a centrifuge. Inthe centrifuge the whole blood is separated into different fractionsdepending upon the density of that fraction. The centrifugal forceseparates the blood sample into different fractions. In addition,various elements may be added to the test tube to create more than twofractions. In particular, commonly used gels may be used to divide thewhole blood into a plurality of different fractions which may includefractions such as platelets, red blood cells, and plasma. Various otherbiological fluids may be separated as well. For example, nucleated cellsmay be separated and extracted from bone marrow or adipose tissuesample.

Many of these systems, however, do not provide a simple or efficientmethod to extract any more than one fraction and especially a fractionother than the top fraction. The top fraction of whole blood is plasma,or other blood constituents suspended in plasma. Thus, to extract otherfractions the plasma fraction must be removed and spun again to obtainthe constituents suspended in this plasma. It is difficult to pierce thetop fraction without co-mingling the sample. Accordingly, obtaining theother fractions is difficult with commonly known systems.

Other systems have attempted to alleviate this problem by providing afloat or other device that is disposed within the sample at theinterfaces of the different fractions during the centrifuge process.Nevertheless, these systems still do not allow a simple way to removethe different fractions without remixing the sample fractions. Inaddition, many of the systems do not allow an easy and reproduciblemethod to remove the desired sample fraction.

Therefore, it is desired to provide a device to allow for the easy andreproducible removal of a particular fraction which does not happen tobe the top fraction of a sample. It is desired to remove the requiredsample without mixing the different fractions during the extractionprocess. In addition, it is desired to provide a device which allows fora consistent extraction which includes known volumes or concentration ofthe fraction elements. Moreover, it is desired to separate andconcentrate a selected fraction with one centrifugation step.

SUMMARY

An apparatus that separates and concentrates a selected fraction orcomponent of a fluid, such as a biological fluid. For example, a buffycoat or platelet fraction or component of a whole blood sample or anundifferentiated cell component of bone marrow or adipose tissue sample.The apparatus, when used with a centrifuge, is generally able to createat least two fractions. It also provides for a new method of extractingthe buffy coat fraction or component or middle fraction from a sample.

The apparatus includes a container to be placed in a centrifuge afterbeing filled with a sample. A buoy or fraction separator, having aselected density that may be less than one fraction but greater than asecond fraction, is disposed in the container. In addition, a secondbuoy may be placed in the container with the first. During thecentrifuge processing, the buoy is forced away from a bottom of thecontainer as the denser fraction collects at the bottom of thecontainer. The buoy is generally able to physically separate the denserfraction from another fraction of the sample.

In addition to providing a first buoy and/or a second buoy, a buoysystem may be provided. Generally, the buoy system may separate thesample into at least three fractions. The fractions may be separated orextracted from the container without substantially commingling thevarious fractions. Generally, a first buoy and a second buoy operatetogether to separate the sample into the various fractions and a syringeor tube may then be interconnected with a portion of the buoy system toextract the selected fractions. For example, a first buoy may begenerally density tuned to a red blood cell fraction of a whole bloodsample, and a second buoy tuned to a density less than the density ofthe plasma fraction.

According to various embodiments a method of forming an enrichedscaffold for application relative to an anatomy is taught. The methodmay include obtaining a volume of a first whole material and obtaining avolume of a second whole material. A first fraction of the first wholematerial and a second fraction of the second whole material may beformed. At least one of the first fraction or the second fraction may beapplied to the scaffold.

According to various embodiments a method of withdrawing a materialdirectly from a patient and collecting a selected fraction of thematerial in a container is taught. The method may include forming anaccess to port to the patient. A pressure differential in a collectioncontainer may be formed relative to the patient. A connection may bemade between the patient and the collection container via the port. Thecollection container may be filled with the material and separating thematerial to form the selected fraction.

Further areas of applicability of the present invention will becomeapparent from the detailed description provided hereinafter. It shouldbe understood that the detailed description and specific examples, whileindicating various embodiment of the invention, are intended forpurposes of illustration only and are not intended to limit the scope ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description and the accompanying drawings, wherein:

FIG. 1 is a plan view of a separator including a depth gage affixed to aplunger in a tube according to a first embodiment of the presentinvention;

FIG. 2 is a cross-section view taken along line 2-2 of FIG. 1;

FIG. 3 is an exploded view of the separator apparatus;

FIG. 4 is a kit including the separator according to an embodiment ofthe present invention;

FIG. 5A is a plan view of the separator being filled;

FIG. 5B is a plan view of a blood sample in the separator after thecentrifuge process;

FIG. 5C is a plan view of the plunger plunged into the tube with thedepth gage to further separate the blood sample;

FIG. 5D is a plan view of the buffy coat and the plasma fractions beingextracted from the separator;

FIG. 6A is a side plan view of a buoy system according to variousembodiments;

FIG. 6B is a cross-sectional view of the buoy system of FIG. 6A;

FIG. 7A is a plan view of a separator according to various embodimentsbeing filled;

FIG. 7B is a plan view of a separator, according to various embodiments,after a centrifugation process;

FIG. 7C is a plan view of a separator system being used to extract aselected fraction after the centrifugation process;

FIG. 7D is a plan view of a second fraction being extracted from theseparator according to various embodiments;

FIG. 8 is a schematic view of an assisted blood withdrawal device; and

FIG. 9 is a block diagram of a method for implanting selected fractionsof a fluid.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

The following description of various embodiments is merely exemplary innature and is in no way intended to limit the invention, itsapplication, or uses. Although the following description exemplaryrefers to a blood separation, it will be understood that the presentinvention may be used to separate and concentrate any appropriatematerial. It will be further understood that many multi-component ormulti-fraction fluids may be separated. The components or fractions aregenerally inter-mingled in the whole sample but may be separated with acentrifuge device that causes increased local gravity or gravitationalforces.

With reference to FIGS. 1-3, according to various embodiments aseparator 10, also referred to as a concentrator, is illustratedaccording to a first embodiment of the present invention. The separator10 generally includes a tube or container 12 that is adapted to hold afluid sample, such as an anti-coagulated whole blood sample, for furtherprocessing. It will be understood that the tube may hold other solutionsincluding constituents of more than one density, such as bone marrow ora mixture of whole blood and bone marrow. The tube 12 includes a top oropen end 12 a, which is closeable, and a bottom or closed end 12 b. Thebottom 12 b may also be selectively closeable.

Disposed within the tube 12 is a first piston or buoy 14 that is able tomove along a central axis A of the tube 12. The buoy 14 is generallynearer the bottom end 12 b of the tube 12 rather than the open end 12 a.Also disposed within the tube 12 is a second piston or plunger 16. Theplunger 16 is also able to move within the tube 12 generally between aposition closer to the open end 12 a to a position closer to the closedend 12 b of the tube 12. A cap 18 substantially mates with the open end12 a of the tube 12 to close the tube 12 save for ports formed in thecap 18. Extending from the cap 18 is a plasma valve or port 20 thatcommunicates with an area, described further herein, within the tube 12defined between the plunger 16 and the cap 18. It will be understoodthat the plasma port 20 is merely exemplary in nature and simply allowsfor removal of a selected fraction of a sample, such as plasma fromwhole blood.

The cap 18 also includes a depth gage port 19. Extending from theplunger 16 and through the depth gage port 19 is a first plunger port22. A depth guide or gage 24 includes a female connector 26 adapted toconnect with the first plunger port 22. The depth gage 24 also includesa depth gage housing or cannula 28. The depth gage housing 28 defines adepth gage bore 30. Incorporated in the housing 28 and extending distalfrom the end mating with the plunger is a neck 32. The neck 32 includesexternal neck threads 34. The external neck threads 34 are adapted toengage appropriate internal threads of a mating member.

The mating member may include a compression nut 36 that mates with theexternal neck threads 34 to lock a depth gage rod 38 in a predeterminedposition. A split bushing 39 is also provided to substantially seal thedepth gage housing 28 when the depth gage rod 38 is locked in place. Thedepth gage rod 38 extends through the depth gage housing 28 andterminates at a rod handle 40. The rod handle 40 may be a form easilymanipulated by a human operator. The rod 38 extends coaxially with axisA of the tube 12. The depth gage rod 38 extends through the plunger 16 apredetermined distance and may be locked at that distance with thecompression nut 36.

Although the tube 12 is described here as a cylinder, it will beunderstood that other shapes may be used, such as polygons. The internalportions, such as the cap 18, buoy 14, and plunger 16, would alsoinclude this alternate shape. Preferably the tube 12 is formed of athermal plastic material which is flexible under the forces required toseparate blood. The tube 12 may be made of a material that includes theproperties of both lipid and alcohol resistance. These properties helpincrease the separation speed and decrease the amount of material whichmay cling to the tube wall 42. For example, Cyrolite MED2® produced byCyro Industries of Rockaway, N.J. may be used to produce the tube 12.

The tube 12 has a tube wall 42 with a thickness of between about 0.01millimeters and about 30.0 millimeters, although the tube wall 42 may beany appropriate thickness. The thickness of the tube wall 42 allows thetube wall 42 to flex during the centrifuge process yet be rigid enoughfor further processing of a blood sample disposed in the tube 12. Thetube 12 is closed at the bottom end 12 b with a tube bottom 44 formed ofthe same material as the tube wall 42 and is formed integrallytherewith. Generally the tube bottom 44 has a thickness which issubstantially rigid under the forces required to separate the samplesuch that it does not flex.

The buoy 14 includes an upper or collection face 46 that defines aninverse cone or concave surface. Generally the cone has an angle ofbetween about 0.5° and about 45°, wherein the apex of the cone is withinthe buoy 14. The collection face 46 forms a depression in the buoy 14which collects and concentrates material during the separation process.Additionally, the buoy 14 has a bottom face 48 that defines an inversecone, dome, or covered surface. The buoy bottom face 48 includes an apex50 that engages the tube bottom 44 before a buoy edge 52 engages thetube bottom 44. The buoy 14 includes a material that is a substantiallyrigid such that the buoy edges 52 never meet the tube bottom 44.Therefore, there is a gap or free space 54 formed between the buoy edge52 and the tube bottom 44 along the perimeter of the buoy 14.

The separator 10 is generally provided to separate a multi-componentfluid that generally includes various components or constituents ofvarying densities that are co-mingled or mixed together. The separator10 includes the buoy 14 that is of a selected density depending upon aselected constituent of the multi-constituent liquid. Although the buoy14 may be tuned or of any selected density, the following examplerelates to separation of whole blood to various components. Therefore,the buoy 14 will be discussed to include a selected density relative towhole blood separation. It will be understood, however, that the buoy 14may be of any appropriate density depending upon the multi-componentfluid being separated.

The buoy 14 may be formed of any appropriate material that may have aselected density. For example, when the separator 10 is to separateblood, the buoy 14 generally has a density which is greater than that ofred blood cells in a whole blood sample, but less than the plasma ornon-red blood cell fraction of a whole blood sample. For blood, thedensity of the buoy 14 is generally between about 1.02 g/cc and about1.09 g/cc.

To achieve the selected density, the buoy 14 may be formed as acomposite or multi-piece construction, including a plurality ofmaterials. Particularly, a first or outside portion 56 defines thecollection face or surface 46 and the buoy edge 52 and is formed of thesame material as the tube 12. The outside portion 56 defines a cup orvoid into which a plug or insert 58 is placed. The insert 58 has a masssuch that the density of the entire buoy 14 is within the selectedrange, for example the range described above. Generally, a high densitypolyethylene may be used, but the material and size of the insert 58 maybe altered to produce the desired density of the buoy 14. Alternatively,the buoy 14 may be formed of a single suitable material that has adensity in the selected range. Nevertheless, the buoy 14 formedunitarily or of a single material would still include the other portionsdescribed in conjunction with the buoy 14.

The outside portion 56 of the buoy 14 also defines the outsidecircumference of the buoy 14. The outside circumference of the buoy 14is very close to the internal circumference of the tube 12. Due to theoperation of the buoy 14, however, described further herein, there is aslight gap between the outside of the buoy 14 and the inside of the tube12. Generally, this gap is between about 1 and about 10 thousandths ofan inch around the entire circumference of the buoy 14. Generally, it isdesired that the distance between the outside circumference of the buoy14 and the inside circumference of the tube 12 is great enough to allowa selected material or component to pass. For example, in whole bloodthe distance is selected so that red blood cells may pass through thegap without being lysed, damaged, or activated.

The plunger 16 includes a plunger front or collection face 60 and aplunger wall 62 that extends from the plunger front face 60. The plungerwall 62 extends relatively perpendicular to the plunger front face 60and substantially parallel to the tube wall 42. Extending from thecenter of the plunger 16 is a sample collection projection 64. Extendingfrom the top of the collection projection 64 is the first plunger port22. The sample collection projection 64 includes a plunger samplecollection bore 68 defined therethrough. The plunger sample collectionbore 68 terminates at a sample collection aperture 70 that issubstantially in the center of the plunger front face 60. The plungerfront face 60 also defines an inverse cone where the sample collectionaperture 70 is the apex of the cone. The plunger front face 60 defines acone with an angle substantially similar or complimentary to thecollection face 46 of the buoy 14. In this way, the plunger front face60 may mate substantially completely with the collection face 46 forreasons described more fully herein.

The plunger 16 also includes a back face 72. Extending from the plungerfront face 60 to the back face 72 is a bore 74. A check valve 76 isoperably connected to the bore 74. The check valve 76 allows a liquid tomove from the plunger front face 60 to the back face 72 while notallowing the liquid to move from the back face 72 to the plunger frontface 60. Therefore, the check valve 76 is substantially a one-way valvewhich allows a material to move in only one direction. The check valve76 may also operate automatically allowing flow in only onepredetermined direction. Alternatively, the check valve 76 may beoperated manually and include a portion extending from the check valve76 requiring manipulation to stop or start a flow through the checkvalve 76.

The plunger 16 may be made out of any appropriate material which doesnot interfere with the separation of the fractions of the fluid, such aswhole blood. The plunger 16, however, is made of a material that isflexible or at least partially deformable. A flexible material allowsthe plunger 16 to have an external circumference defined by the plungerwalls 62 that is substantially equal to the internal circumference ofthe tube 12. Because of the deformability of the plunger 16, however,the plunger 16 is still able to move within the tube 12. The plunger 16is able to move through the tube 12 and also substantially wipe theinterior of the tube wall 42. This creates, generally, a moveable sealwithin the tube 12. Thus, substantially no material escapes the actionof the separator 10 when the plunger 16 is plunged into the tube 12.This also helps concentrate the portion of the sample desired to becollected, described more fully herein.

The cap 18 provides a structure to substantially close the tube 12. Thecap 18 particularly includes a plate 78 that has an externalcircumference substantially equal to the external circumference of thetube 12. Extending from the plate 78 and into the tube 12 is a flange80. The external circumference of the flange 80 is substantially equalto the internal circumference of the tube 12. In this way, the cap 18substantially closes the tube 12. It will be understood the cap 18 maybe in any form so long as the cap 18 substantially closes and/or sealsthe tube 12 when installed.

Formed through the center of the plate 78 is the depth gage port 19. Thedepth gage port 19 is also adapted to receive the sample collectionprojection 64. The first plunger port 22 extends above the plate 78through the depth gage port 19. The circumference of the depth gage port19 is substantially equal to the external circumference of the samplecollection projection 64 such that a liquid seal is formed. The plate 78defines a sample face 84 that includes an interior side of the cap 18.The area between the sample face 84 of the cap 18 and the back face 72of the plunger 16 define a plasma collection area 86. Although theplasma collection area 86 is exemplary called the plasma collectionarea, it will be understood that the plasma collection area 86 may alsocollect any appropriate fraction of the sample that is positioned withina separator 10. The plasma collection area 86 is merely an exemplaryname and an example of what material may be collected in the area of theseparator 10. As discussed herein, the separator 10 may used to separatewhole blood into various fractions, therefore the plasma collection area86 is used to collect plasma. The plasma collection area 86 also allowsa space for the check valve 76 to be installed.

A second bore 88 is formed in the plate 78. Extending through the secondbore 88 is the plasma collection valve 20. In liquid communication withthe plasma collection valve 20 is a plasma collection tube 92. Theplasma collection tube 92 has a length such that the plasma collectiontube 92 is able to extend from the plasma collection valve 20 tosubstantially the tube bottom 44. The plasma collection tube 92,however, is flexible enough such that it may be folded or compressed tofit within the plasma collection area 86 when the plunger issubstantially near the top 12 a of the tube 12. The plasma collectiontube 92 may also be connected to a hose barb 93 that includes a plasmacollection bore 93 a. The plasma collection bore 93 a is substantiallylevel with the plunger back face 72. Alternatively, the plasmacollection bore 93 a may be positioned below the plunger back face 72but in fluid communication with the plasma collection tube 92.

The outboard side of the plasma collection valve 20 may include externalthreads 94 to mate with internal threads of a plasma valve cap 96.Therefore, the plasma collection valve 20 may be selectively opened andclosed via the plasma valve cap 96. It will be understood, however, thatother appropriate means may be used to open and close the plasmacollection valve 20 such as a clip or a plug. It will be understood thatthe plasma collection valve 20, plasma collection tube 92, plasmacollection bore 23 a may be used to collect any appropriate material orfraction from the separator 10.

Also formed in the plate 78 is a vent bore 98. The vent bore 98 allowsair to flow into the collection area 86 as the plunger 16 is beingplunged into the tube 12. The vent bore 98 may include a filter 100 suchthat liquid cannot escape from the tube 12. The filter 100 allows air toenter or escape from the collection area 86 while maintaining the liquidseal of the tube 12 produced by the cap 18.

Selectively attachable to the first plunger port 22 is the depth gage24. The female connector 26 interconnects the depth gage housing 28 tothe first plunger port 22. Internal threads in the female connector 26mate with an external thread 102 formed on the first plunger port 22. Itwill be understood, however, that other engagement mechanisms betweenthe depth gage 24 and the plunger 16 may be used. For example, a snapconnection rather than a threaded connection between the two may beused.

The depth gage housing 28 is formed to be substantially rigid. Suitablematerials, when sized properly, include polycarbonate and CYRO MED2®.The material preferably is both rigid and does not substantially reactwith the sample. It is rigid enough to provide a mechanism to plunge theplunger 16 into the tube 12. In addition the external circumference ofthe depth gage housing 28 is substantially equal to the circumference ofthe depth gage port 19 in the plate 78. Therefore, as the plunger 16 isbeing plunged into the tube 12 with the depth gage 24, no liquidmaterial is allowed to escape around the depth gage housing 28 andthrough depth gage port 19.

Formed within the depth gage housing 28 is the bore 30 which receivesthe depth gage rod 38. The depth gage rod 38 extends through the samplecollection bore 68 of the sample collection projection 64 and protrudesthrough the sample collection aperture 70 a predetermined length. Thedepth gage rod 38 extends through the sample collection aperture 70 alength such that when an end 104 of the depth gage rod 38 meets the buoy14, the volume defined by the collection face 46 and the plunger frontface 60 is between about 5 percent and about 30 percent of the totalvolume of the sample that the tube 12 holds. The projection of the depthgage rod 38 allows for an easily reproducible collection amount andconcentration over several trials.

The compression nut 36 locks the depth gage rod 38 in the predeterminedposition. Nevertheless, once the plunger 16 has been plunged to thedesired depth in the tube 12, the compression nut 36 may be loosened sothat the depth gage rod 38 may be removed from the plunger 16 and thedepth gage housing 28 without moving the plunger 16. A syringe or otherappropriate device may then be affixed to the external neck threads 34of the depth gage 24 to extract the fraction or phase that is betweenthe plunger front face 60 and the collection face 46. As describedfurther herein, the fraction or phase that is left between the plungerfront face 60 and the collection face 46 may be the buffy coat of awhole blood sample. Nevertheless, it will be understood that thefraction between the plunger front face 60 and the collection face 46may be any appropriate fraction of the sample that is disposed in theseparator 10.

The separator 10 may be provided alone or in a kit 200, as illustratedin FIG. 4. The kit 200 may be placed in a tray 202 which is covered toprovide a clean or sterile environment for the contents of the kit 200.The kit 200 may include at least a first separator 10 and a secondseparator 10′. A first depth gage 24 and a second depth gage 24′ arealso provided, one for each separator 10, 10′. The kit 200 alsogenerally includes a first syringe 204, including a needle, to draw abiological sample, such as blood from a patient. The first syringe 204may also be used to place the sample in the first separator 10. Aftercentrifuging the sample a second device or syringe 210 may be used toextract a first fraction of the sample. While a third device or syringe212 may be used to extract a second fraction of the sample. Also atourniquet 214 and other medical supplies, such as gauze 216 and tape218, may be provided to assist the practitioner. It will be understoodthe elements of the kit 200 are merely exemplary and other appropriateitems or elements may be included.

With reference to FIGS. 5A-5D a method using the blood separator 10 isillustrated. The following example relates specifically to the takingand separation of a sample of whole blood from a patient. Nevertheless,it will be understood that another appropriate biological material maybe separated and concentrated using the separator 10. For example, bonemarrow may be separated and concentrated using the separator 10. Thevarious fractions of the bone marrow are similar to the fractions ofwhole blood. Generally, the bone marrow includes a fraction thatincludes substantially dense material and a second phase that is lessdense and has other components suspended therein, such as nucleatedcells. The bone marrow sample may be positioned in the separator 10,similarly to the whole blood as described herein, and separated in asubstantially similar manner as the whole blood. The separator 10 canthen be used to remove nucleated cells from the bone marrow samplewhereas the separator 10, as described herein, is used to remove thebuffy coat from the whole blood which includes platelets and otherappropriate materials.

A mixture of whole blood and bone marrow may be positioned in theseparator 10 for separation and concentration. Similar methods and stepswill be used to separate the mixture of whole blood and bone marrow witha main difference being the material that is separated. It will also beunderstood that various centrifuge times or forces may be altereddepending upon the exact material that is being separated with theseparator 10. It will also be understood that the separation of wholeblood, bone marrow, or a mixture of whole blood and bone marrow aremerely exemplary of the materials that may be separated using theseparator 10.

With reference to FIGS. 5A-5D and to a whole blood sample, a sample ofwhole blood taken from a patient is placed in the tube 12 with ananticoagulant using the first syringe 204 or other appropriate deliverymethod. In particular, the first syringe 204 may be connected to thefirst plunger port 22, after which the blood sample is provided to thetube 12 via the sample collection bore 68 and sample collection aperture70. A cap 220 is then placed over the first plunger port 22 tosubstantially seal the tube 12.

After the whole blood sample is delivered to the tube 12, the separator10 is placed in a centrifuge. The second separator 10′, substantiallyidentical to the first, is placed opposite the first separator 10including the sample in a centrifuge. The second separator 10′ may alsoinclude a second sample or may include a blank, such as water, so thatthe centrifuge is balanced. The second separator 10′ balances thecentrifuge, both by weight and dynamics.

The separator 10 is then spun in the centrifuge in a range between about1,000 and about 8,000 RPMs. This produces a force between about 65 andabout 4500 times greater than the force of normal gravity, as generallycalculated in the art, on the separator 10 and the blood sample placedin the separator 10. At this force, the more dense material in a wholeblood sample is forced towards the bottom 12 b of the tube 12. The densematerial, such as red blood cells or a red blood cell fraction 222,collects on the tube bottom 44. Because the buoy 14 has a density thatis less than the red blood cell fraction 222, it is forced in adirection toward the top 12 a of the tube 12 in the centrifuge.Nevertheless, because the buoy 14 is denser than a plasma fraction 224,the buoy 14 does not reach the top 12 a of the tube 12.

The forces also affect the tube wall 42. The forces compress the tube 12linearly along axis A thereby bowing or flexing the tube wall 42. As thetube wall 42 compresses it increases the diameter of the tube 12 makingit easier for the buoy 14 to move in the direction of the top 12 a ofthe tube 12. In addition, the bottom face 48, defining an inverse cone,helps the initial movement of the buoy 14. Because the buoy 14 is notsubstantially flat along its bottom, it does not form a vacuuminteraction with the tube bottom 44. Therefore, the initial movement ofthe buoy 14 away from the tube bottom 44 is quicker than if the bottomof the buoy 14 was flat.

During the centrifuge process the red bloods cells of the red blood cellfraction 222 force the buoy 14 in the direction of the top 12 a of thetube 12 because the buoy 14 is less dense than the red blood cellfraction 222. Although the whole blood sample, including the red bloodcells is loaded above the buoy 14, the red blood cells are able to movebetween the buoy 14 and the tube wall 42 because the circumference ofthe buoy 14 is less than the internal circumference of the tube 12.During the centrifuge process the buoy 14 stops at an interface of aplasma fraction 224 and the red blood cell fraction 222 because of theselected or tuned density of the buoy 14.

With particular reference to FIG. 5B, the centrifuge process has beencompleted and the buoy 14 has moved to the interface of the red bloodcell fraction 222 and plasma fraction 224. After the tube 12 has beenremoved from the centrifuge, the tube wall 42 decompresses which helpssupport the buoy 14 at the interface position. It is also understoodthat applying an external pressure to the tube 12 via fingers or anotherapparatus may help stabilize the buoy 14 during the plunging proceduredescribed herein.

On or near collection face 46 is a third fraction 226 including a small,yet concentrated, amount of red blood cells, white blood cells,platelets, and a substantial portion of a buffy coat of the bloodsample. Although the plasma is also present near the collection face 46at this point the solid portions of the buffy coat are more compressedagainst the collection face 46. The position of the buoy 14 also helpsin this matter. Because the buoy 14 is a single body it defines theinterface of the plasma traction 224 and the red blood cell fraction222. Also the density of the buoy 14 assures that it has not passed intothe plasma fraction 224. Therefore, the fractions remain separated afterthe centrifuge process. In addition because the buoy 14 is tuned to thedensity of the red blood cell fraction 222, it is not affected byvariations in the density of the plasma fraction 224 and the buoy's 14position is always at the interface of the red blood cell fraction 222and the plasma fraction 224.

With particular reference to FIG. 5C, the depth gage 24 is affixed tothe first plunger port 22 of the sample collection projection 64. Afterconnecting the depth gage 24 to the first plunger port 22, the plunger16 is plunged into the tube 12 by pushing on the depth gage 24. As thisis performed the plasma fraction 224, formed and separated above thebuoy 14, is able to flow through the check valve 76 into the plasmacollection area 86. This displacement of the plasma fraction 224 allowsthe plunger 16 to be plunged into the tube 12 containing the bloodsample.

The plunger 16 is plunged into the tube 12 until the point where the end104 of the depth gage rod 38 reaches the buoy 14. The volume left in thecollection face 46 is the third fraction 226 and is determined by thedepth gage 24. It may be adjusted by selectively determining the amountthat the depth gage rod 38 extends below the plunger front face 60. Byadjusting the depth gage 24, the concentration of the third fraction 226can be adjusted depending upon the desires of the operator.

The plasma fraction 224 is held in the plasma collection area 86 forlater withdrawal. Therefore, the use of the plunger 16 and the buoy 14creates three distinct fractions that may be removed from the tube 12after only one spin procedure. The fractions include the red blood cellfraction 222, held between the buoy 14 and the tube bottom 44. The thirdor buffy coat fraction 226 is held between the plunger 16 and the buoy14. Finally, the plasma fraction 224 is collected in the plasmacollection area 86.

The third fraction 226 may be extracted from the tube 12 first, withoutcommingling the other fractions, through the sample collection bore 68.With particular reference to FIG. 5D, the depth gage rod 38 may beremoved from the depth gage housing 28. This creates a sample collectioncannula which includes the depth gage bore 30, the sample collectionbore 68, and the sample collection aperture 70. After the depth gage rod38 has been removed, the second syringe 210 may be affixed to the depthgage housing 28 via the external neck threads 34. The second syringe 210may be substantially similar to the first syringe 204.

Before attempting to withdraw the third fraction 226 the separator 10may be agitated to re-suspend of the platelets and concentrated redblood cells in a portion of the plasma remaining in the collection face46. This allows for easier and more complete removal of the thirdfraction 226 because it is suspended rather than compressed against thecollection face 46. A vacuum is then created in the second syringe 210by pulling back the plunger to draw the third fraction 226 into thesecond syringe 210.

As the third fraction 226 is drawn into the second syringe 210 theplunger 16 moves towards the buoy 14. This action is allowed because ofthe vent bore 98 formed in the cap 18. Atmospheric air is transferred tothe plasma collection area 86 through the vent bore 98 to allow thethird fraction 226 to be removed. This also allows the movement of theplunger 16 towards the buoy 14. This action also allows the plunger 16to “wipe” the collection face 46. As the plunger front face 60 mateswith the collection area 46 the third fraction 226 is pushed into thesample collection aperture 70. This ensures that substantially theentire third fraction 226 collected in the collection area 46 is removedinto the second syringe 210. It also increases the consistency of thecollection volumes. In addition, because the second syringe 210 does notprotrude out the sample collection aperture 70, it does not interferewith the collection of the third fraction 226. Once the plunger frontface 60 has mated with the collection face 46 there is substantially novolume between the plunger 16 and the buoy 14.

Once the third fraction 226 is extracted the second syringe 210 isremoved from the first plunger port 22. Also the extraction of the thirdfraction 226 leaves the plasma fraction 224 and the red blood cellfractions 222 separated in the tube 12. At this point a third syringe212 may be affixed to the plasma collection valve 20. The third syringe212 is connected to the external threads 94 of the plasma collectionvalve 20 to ensure a liquid tight connection. It will be understood,however, that another connection mechanism such as a snap or compressionengagement may be used to connect the third syringe 212 to the plasmacollection valve 20.

A vacuum is then created in the third syringe 212 to draw the plasmafraction 224 from the plasma collection area 86 through the plasmacollection tube 92. As discussed above, the plasma collection tube 92 isconnected to the hose barb 93. Therefore, the plasma flows through theplasma collection bore 93 a through the hose barb 93, and then throughthe plasma collection tube 92. It will be understood that the plasmacollection tube 92 may alternatively simply rest on the plunger backface 72 to collect the plasma fraction 224. In this way the plasmafraction 224 may be removed from the blood separator 10 withoutcommingling it with the red blood cell fraction 222. After the plasmafraction 224 is removed, the separator 10 may be dismantled to removethe red blood cell fraction 222. Alternatively, the separator 10 may bediscarded in an appropriate manner while retaining the red blood cellfraction 222.

The separator 10 allows for the collection of three of a whole bloodsample's fractions with only one centrifugation spin. The interaction ofthe buoy 14 and the plunger 16 allows a collection of at least 40% ofthe available buffy coat in the whole blood sample after a centrifugeprocessing time of about 5 minutes to about 15 minutes. Thecomplimentary geometry of the plunger front face 60 and the collectionface 46 help increase the collection efficiency. Although only the conegeometry is discussed herein, it will be understood that various othergeometries may be used with similar results.

The plunger front face 60 being flexible also helps ensure a completemating with the collection face 46. This, in turn, helps ensure thatsubstantially the entire volume between the two is evacuated. Theprocess first begins with the suction withdrawal of the third fraction226 via the second syringe 210, but is completed with a fluid forceaction of the third fraction 226 as the plunger front face 60 mates withthe collection face 46. As the plunger front face 60 mates with thecollection face 46 the fluid force assists in removal of the selectedfraction.

The plunger 16 also substantially wipes the tube wall 42. Because theplunger 16 is formed of a flexible material it forms a seal with thetube wall 42 which is movable. Therefore, substantially no liquid isable to move between the plunger wall 62 and the tube wall 42. Materialis substantially only able to go past the plunger front face 60 via thecheck valve 76.

The complimentary geometry also helps decrease the collection time ofthe third fraction 226. Therefore, entire time to prepare and remove thethird fraction 226 is generally about 5 to about 40 minutes. Efficiencyis also found because the separator 10 allows for the removal of thethird fraction 226 without first removing the plasma fraction 224, whichincludes the buffy coat, and respinning the plasma fraction 224. Onespin in the separator 10 with the whole blood sample allows for theseparation of the buffy coat for easy extraction through the plunger 16.

As discussed above, the separator 10 may be used to separate anyappropriate multi-component material. For example, a bone marrow samplemay be placed in the separator 10 to be centrifuged and separated usingthe separator 10. The bone marrow sample may include several fractionsor components that are similar to whole blood fractions or may differtherefrom. Therefore, the buoy 14 may be altered to include a selecteddensity that is dependent upon a density of a selected fraction of thebone marrow. The bone marrow may include a selected fraction that has adifferent density than another fraction and the buoy 14 may be designedto move to an interface between the two fractions to allow for aphysical separation thereof. Similar to the whole blood fraction, theplunger 16 may then be moved to near a collection face 46 of the buoy14. The fraction that is then defined by the collection face 46 and theplunger 16 may be withdrawn, as described for the removal of the buffycoat from the whole blood sample. For example, the middle fraction orthird fraction in the bone marrow sample may include a fraction ofundifferentiated or stem cells.

It will also be understood that mixtures of various fluids may beseparated in the separator 10. For example, a mixture of whole blood andbone marrow may be positioned in the separator 10 at a single time. Thebuoy 14 may be tuned to move to an interface that will allow for easyremoval of both the buffy coat, from the whole blood sample, and theundifferentiated cells, from the bone marrow sample. Nevertheless, itwill be understood that the separator 10 may be used within anyappropriate biological material or other material having multiplefractions or components therein. Simply, the buoy 14 may be tuned to theappropriate density and the plunger 16 may be used to cooperate with thebuoy 14 to remove a selected fraction.

With reference to FIGS. 6A and 6B, a buoy system 300 is illustrated. Thebuoy system 300 generally includes a first buoy or fraction separatormember 302 and a second buoy member or fraction separator 304. The firstbuoy 302 and the second buoy 304 may be operably interconnected with abuoy system cylinder or member 306. The buoy system 300 may be placed ina tube, such as the tube 12. The tube 12 may be formed of anyappropriate material, such as the Cryolite Med®2 as discussed above.Nevertheless, the buoy system 300 may be designed to fit in the tube 12or may be formed to fit in any appropriate member that may be disposedwithin a selected centrifuging device. It will be understood that thefollowing discussion relating to buoy system 300 to be substantiallymatched to the size of the tube 12 is merely exemplary. As the buoy 14may be sized to fit in any appropriate tube, the buoy system 300 mayalso be sized to fit in any appropriate tube. It will be furtherunderstood that the tube 12 may be any appropriate shape. The tube 12need not only be cylindrical but may also be or include conicalportions, polygonal portions, or any other appropriate shapes.

The first buoy 302 of the buoy system 300 may be generally similar ingeometry to the buoy 14. It will be understood that the first buoymember 302 may be formed in the appropriate manner including shape orsize to achieve selected results. Nevertheless, the first buoy member302 generally includes an exterior diameter that may be slightly smallerthan the interior diameter of the tube 12. Therefore, the first buoymember 302 may be able to move within the tube 12 during the centrifugalprocess. Also, as discussed above, the tube 12 may flex slightly duringthe centrifuging process, thus allowing the first buoy member 302 toinclude an exterior diameter substantially equivalent to the interiordiameter of the tube 12. As discussed further herein, during thecentrifugation process, a portion of the fraction of a sample may passbetween the exterior wall of the first buoy member 302 and the tube 12.

The first buoy member 302 may generally include a density that issubstantially equivalent to a first or selected fraction of the sample.If the sample to be separated includes whole blood and is desired toseparate the red blood cells from the other portions of the sample, thefirst buoy member 302 may have a selected density that may be about 1.00grams per cc (g/cc) to about 1.10 g/cc. It will be understood that thedensity of the first buoy member 302 may be any appropriate density,depending upon the fraction to be separated, and this range of densitiesis merely exemplary for separating red blood cells from a whole bloodsample.

In addition, the first buoy member 302 includes a collection face orarea 308 at a proximal or upper portion of the first buoy member 302.The collection face 308 generally defines a concave area of the firstbuoy member 302 and may have a selected angle of concavity. The buoyassembly 300 defines a central axis D. The collection face 308 defines asurface E that is formed at an angle γ to the central axis D of the buoysystem 300. The angle γ may be any appropriate angle and may be about0.5° to about 45°. Nevertheless, it will be understood that the angle γmay be any appropriate angle to assist in collection of a selectedfraction or portion of the sample by the first buoy member 302.

A bottom or lower surface 310 of the first buoy member 302 may define abottom face. The bottom face 310 may also be formed at an angle Drelative to the central axis D. The bottom surface 310 defines a surfaceor plane F that may be formed at an angle Δ relative to the central axisD of the buoy system 300. The angle Δ may be any appropriate angle andmay be about 0.5° to about 45°. Similarly to the buoy bottom face 48,the bottom surface 310 defines an apex 312 that may first engage thebottom 12 d of the tube 12, such that most or the majority of the bottomsurface 310 does not engage the tube 12. As illustrated further herein,the apex 312 allows for a free space or gap to be formed between thebottom face 310 of the first buoy member 302 and the bottom 12 b of thetube 12.

The second buoy member 304 may include an outer diameter substantiallyequivalent to the outer diameter of the first buoy member 302.Therefore, the second buoy 304 may move with the first buoy 302,particularly if the second buoy 304 is interconnected with the firstbuoy 302 with the buoy central cylinder 306. Nevertheless, the secondbuoy member 304 may be allowed to move substantially freely within thetube 12 during the centrifuging process.

The second buoy member 304 also includes an upper or superior surface314 that defines a plane G that is formed at an angle relative to thecentral axis D of the buoy system 300. The angle ε of the plane Grelative to the central axis D of the buoy system 300 may be anyappropriate angle. For example, the angle ε may be about 90° to about150°. Generally, the angle ε may assist in allowing a selected fractionor a portion of the sample to pass over the top surface 314 and past thesecond buoy member 304 during the centrifuging process.

The second buoy member 304 also define a bottom or inferior surface 316that also defines a plane H that may be formed at an angle K relative tothe central axis D of the buoy system 300. The angle K may be anyappropriate angle, such as about 90° to about 150°. Nevertheless, theangle K may be substantially complimentary to the angle γ of thecollection face 308 of the first buoy member 302. For example, if theangle γ is about 80°, the angle K may be about 100°, such thatsubstantially 180° or a straight line is formed when the first buoymember 302 engages the second buoy member 304. This may be for anyappropriate reason, such as extraction of a fraction that may bedisposed near the collection face 308 of the first buoy member 302.Nevertheless, the angle K may be any appropriate angle as the angle γ.

The second buoy member 304 may be formed to include any appropriatedensity. For example, the second buoy member 304 may include a densitythat is less than the plasma fraction of a whole blood sample. It willbe understood that the second buoy member 304 may include anyappropriate density and a density that is less than the plasma fractionof a whole blood sample is merely exemplary. Nevertheless, if a wholeblood sample is desired to be separated and the plasma sample is to besubstantially separated from another fraction, the second buoy member304 may include a density that is less than the plasma fraction of thewhole blood sample. As described herein, if the second buoy member 304includes a density less than the plasma fraction of a whole blood sampleand the first buoy member 302 includes a density greater than that ofthe red blood cells, the buoy system 300 may be substantially positionednear an interface between the red blood cell fraction and the plasmafraction of a whole blood sample. Therefore, as discussed above, andfurther described herein, the platelet or buffy coat fraction of thewhole blood sample may be substantially collected near or in thecollection face 308 of the buoy system 300.

The buoy post 306 may operably interconnect the first buoy member 302and the second buoy member 304. The buoy post 306 may be any appropriateconnection member. The buoy post need not be a single cylindricalportion. For example the buoy post 306 may include one or more membersinterconnecting the first buoy member 302 and the second buoy member304, such as around a perimeter thereof. In addition, the buoy post 306may include any appropriate shape or geometry.

The buoy system post 306 may be rigidly affixed to the first buoy member302 and the second buoy member 304, such that the first buoy member 302may not move relative to the second buoy member 304 and vice versa.Alternatively, the buoy post 306 may be slidably connected to either orboth the first buoy member 302 and the second buoy member 304. Accordingto various embodiments, the buoy post 306 is generally fixedly connectedto the first buoy member 302 and slidably interconnected to the secondbuoy member 304. The buoy post 306 may include a catch portion or lip320 that is able to engage a portion of the second buoy member 304, suchthat a range of travel of the second buoy member 304, relative to thefirst buoy member 302 is limited. Nevertheless, the range of travel ofthe second buoy member 304 towards the first buoy member 302 may besubstantially unlimited until the second buoy member 304 engages thefirst buoy member 302.

The buoy post 306 may also define a central cannula or bore 322. Thepost bore 322 may include a connection portion 324 substantially definednear an upper or a proximal end of the buoy post 306. This may allow forinterconnection of various components with the buoy post 306, such thatvarious components may be moved through the bore 322 from an exteriorlocation. The buoy post 306 may also define a port or cannula 326 thatconnects the post cannula 322 with the collection face 308. Therefore, asubstance may travel through the post cannula 322 and through the port326. Various substances may then be provided to or removed from thecollection face 308 of the first buoy member 302.

The buoy system 300 may be used to separate a selected multi componentsample, such as a whole blood sample. With continuing reference to FIGS.6A and 6B, and reference to FIGS. 7A-7D, a method of using the buoysystem 300, according to various embodiments, is illustrated anddescribed. With reference to FIGS. 7A-7D, like reference numerals areused to indicate like portions of the tube 12 and the associatedmechanisms described in FIGS. 1-3. Therefore, it will be understood thatthe buoy system 300 may be used with the tube 12 or any otherappropriate tube or container system or apparatus. Nevertheless, forsimplicity, the description of a method of use of the buoy system 300will be described in conjunction with the tube 12.

The tube 12 may include the cap 18 that further defines a plasma valveor port 20. Extending through the cap 18 and interconnecting with afirst flexible tube or member 92, the plasma port 20 may be used toextract a selected fraction of the sample that is positioned above thesecond buoy member 304. As illustrated above, the tube 92 may also beinterconnected with a selected portion of the system, such as the topsurface 314 of the second buoy member 304. As illustrated above, a valvemay be positioned and is operably interconnected with the tube 92 withthe upper surface 314 of the second buoy member 304. Nevertheless, sucha valve is not necessary and it may be provided merely for convenience.

Other portions of the blood separator system 20, particularly thoseportions of the tube 12 and the cap 18 that have various valvesconnected therewith may be included in the tube 12 and used with thebuoy system 300. Nevertheless, once the buoy system 300 isinterconnected, it may be positioned in the interior of the tube 12 andthe syringe 204 used to place a sample into the tube 12. The sample maybe expressed from the syringe 204 into the interior of the tube 12 andthe sample may be any appropriate sample, such as a whole blood sample.Nevertheless, it will be understood, such as discussed above, variousother samples may be used, such as bone marrow samples, a mixture ofbone marrow and whole blood or nonbiological fluids or materials. Also,the sample may be placed in the tube 12 according to various methods. Asdescribed above, an anticoagulant or other components may be mixed withthe whole blood sample, if a whole blood sample is used, before thewhole blood sample is positioned within the tube 12. The syringe 204 isconnected with the plunger port 22 extending from the cap 18, although aplunger may not be used in various embodiments.

After the sample is positioned within the tube 12, as described above, acap may be positioned over the port 22, such that the sample is notallowed to escape from the tube 12. After the sample is placed in thetube 12 and the cap placed on the port 22, the tube 12 including thesample and the buoy system 300 may be centrifuged.

With reference to FIG. 7B, after a centrifugation of the tube 12,including the buoy system 300, substantially three fractions of thesample may be formed. A first fraction 330 may be positioned between thebottom face 310 and the bottom of the tube 44. A second fraction may bepositioned between the collection face 308 and the bottom surface 316 ofthe second buoy 304. In addition, a third fraction may be positionedbetween the upper surface 314 and the cap 18 of the tube 12. Generally,the first fraction 330, the second fraction 332, and the third fraction334 are substantially physically separated with the buoy system 300.During the centrifugation process, the tube 12 may flex slightly toallow for ease of movement of the buoy system 300 through the tube 12and the sample. Nevertheless, the buoy system 300, during thecentrifugation process, substantially creates the three fractions 330,332, and 334 without the operation of an operator. Therefore, theformation of at least three fractions may be substantially simultaneousand automatic using the buoy system 300.

The buoy system 300 substantially separates the fractions 330, 332, and334, such that they may be easily removed from the tube 12. For example,with reference to FIG. 7C, a syringe or other instrument 340 may be usedto extract the second fraction 332 by interconnecting a cannula or boredtube 342 with the connection portion 324 of the buoy cylinder 306. Bydrawing the plunger 344 into the extraction syringe 340, a vacuum orupward force is produced within the extraction syringe 340. This forcedraws the second fraction 332 through the ports 326 of the buoy post 306and through the buoy cannula 322. Therefore, the second fraction 332 maybe extracted from the tube 12 without substantially commingling thesecond fraction 332 with either the first fraction 330 or the thirdfraction 334. The second fraction 332 is drawn in the direction of arrowM through the cannula 322 and into the extraction syringe 340.

Alternatively, if the post 306 is not provided other portions may beprovided to gain access to the second fraction 332. For example, if aplurality of members are provided around the perimeter of the first buoy302 and the second buoy 304 a valve portion, such as a puncture-ablevalve, may be provided in the second buoy 304 to be punctured with anobject. In this way an extraction needle may puncture the valve to gainaccess to the second fraction 332. Regardless, it will be understoodthat the buoy system 300 may be able to form a plurality of fractions,such as the three fractions 330, 332, and 334 and at least the secondfraction 332 may be extracted without substantially commingling thevarious fractions.

During the extraction of the second fraction 332 through the cannula322, the second buoy member 304 may move in the direction of arrow Ntowards the first buoy member 302. As described above, the collectionface 308 of the first buoy member may include an angle γ that issubstantially complementary to the bottom face 316 of the second buoymember 304. Therefore, if the second buoy member 304 is allowed to movealong the buoy cylinder 306, the bottom face 316 of the second buoymember 304 may be able to substantially mate with the collection face308 of the first buoy member 302. Alternatively, if the second buoymember 304 is not allowed to move, the second buoy member may beprovided with a vent port or valve, such that the extraction of thesecond fraction 332 from the collection face 308 may not be hindered bythe buildup of undesirable forces. Nevertheless, if the second buoymember 304 may move, the interaction of the bottom face 316 of thesecond buoy member 304 may assist in substantially removing the entiresecond fraction 332 from the tube 12. As described above, the bottomface 60 of the plunger 16 may also serve a similar purpose when engagingthe collection face 46 of the buoy 14.

With reference to FIG. 7D, once the second fraction 332 has beenextracted from the tube 12, the second buoy member 304 may substantiallymate with a portion of the first buoy member 302. As discussed above,the second buoy member 304 may substantially only mate with the firstbuoy member 302 if the second buoy member 304 is able to substantiallymove relative to the first buoy member 302. Therefore, it will beunderstood that the second buoy member 304 need not necessarily matewith the first buoy member 302 and is merely exemplary of an operationof various embodiments. Nevertheless, once the second fraction 332 hasbeen extracted from the tube 12, the port 20 may be used in conjunctionwith a selected instrument, such as a plasma extraction syringe 212 toremove the plasma or the third fraction 334 from the tube 12 using theextraction tube 92 interconnected with the port 20.

As described above, the tube 92 allows for extraction of the thirdfraction 334 from the tube 12 without commingling the third fraction 334with the remaining first fraction 330 in the tube 12. Therefore, similarto the separator and extraction system 10, three fractions may besubstantially formed within the tube 12 with the buoy system 300 and maybe extracted without substantially commingling the various fractions.Once the third fraction 334 is extracted from the tube 12, the buoysystem 300 may be removed from the tube 12, such that the first fraction330 may be removed from the tube 12. Alternatively, the first fraction330 may be discarded with the tube 12 and the buoy system 300 as adisposable system. Alternatively, the system may be substantiallyreusable, such that it can be sterilized and may be sterilized forvarious uses.

The description of the method of use of the buoy system 300 is exemplaryof a method of using a system according to various other embodiments. Itwill be understood, however, that various specifics may be used fromvarious embodiments to allow for the extraction of selected fractions.For example, the centrifugation process may be substantially a singlestep centrifugation process. The buoy system 300, according to variousembodiments, may allow for the formation of three fractions during asingle centrifugation process. This centrifugation process may occur atany appropriate speed, such as about 1000 rpms to about 8000 rpms. Thisspeed may produce a selected gravity that may be approximately 4500times greater than the normal force of gravity. Nevertheless, thesespecifics are not necessary to the operation of the buoy system 300according to various embodiments. The buoy system 300, according tovarious embodiments, may be used to extract a plurality of fractions ofa sample after only a single centrifuging process and withoutsubstantially commingling the various fractions of the sample.

With reference to FIG. 8, the blood collection and separation systemthat includes the tube 12, according to various embodiments, may befilled with a multi-component fluid or solution, such as blood from apatient, is illustrated. The tube 12 may include any appropriateseparation system, such as the separation system 300. Nevertheless, inaddition to filling the tube 12 with a fluid from the syringe 204 anyappropriate method may be used to fill the tube 12. For example, when asolution, including a plurality of components, is placed into the tube12 it may be collected directly from a source.

For example, a patient 350 may be provided. The patient 350 may beprovided for a selected procedure, such as generally an operativeprocedure or other procedure that requires an intravenous connection352, such as a butterfly needle, to be provided in the patient 350. Theintravenous connection 352 generally provides a tube 354 extendingtherefrom. The tube 354 may be used to withdraw fluids from the patient350 or provide materials to the patient 350, such as medicines or otherselected components. Nevertheless, the intravenous connection 352 isgenerally provided for various procedures and may be used to fill thetube 12.

The tube 354 may interconnect with the plunger port 22 or anyappropriate portion of the tube 12. The port 22 may be used to connectwith the tube 354 in a similar manner as it would connect with thesyringe 204, if the syringe 204 was provided. Nevertheless, it will beunderstood that the tube 354 may be provided directly to the tube 12from the patient 350. This may reduce the number of steps required tofill the tube 12 and reduce possible cross-contamination from thepatient 350 with the various components. Moreover, making a connectiondirectly with the patient 350 may make the withdrawal and collection ofblood from the patient 350 more efficient.

Once the tube 354 is interconnected with the tube 12 the pressuredifferential between the patient 350, such as the intravenous pressureof the blood, may be used to fill the tube 12 to a selected volume. Inaddition, a vacuum system 356 may be provided. The vacuum system 356 mayinclude a vacuum inducing portion or member 358, such as a resilientbulb. The vacuum inducing member 358 may be interconnected with the tube12 through a selected connecting portion 360.

The vacuum connecting portion 360 may interconnect with an orifice 362.The orifice 362 may be interconnected or extend from the cap 18 orprovided in any appropriate portion with the tube 12. Nevertheless, afirst one way valve 364 may be provided along the connection portion 360or near the orifice 362. The one way valve 364 provides that a flow of afluid, such as a gas, may pass in a first direction but not in a second.A second one way valve 366 may also be provided downstream from thefirst one way valve 364. In this way, a vacuum may be created with thevacuum inducing member 358, such that air is drawn out of the tube 12and removed through the second one way valve 366 in the direction ofarrow V. Due to the first and second one-way valves 364, 366 the air isgenerally withdrawn from the tube 12 without substantially allowing theair to flow back into the tube 12. Thus, a vacuum can be created withinthe tube 12 to assist with removing a selected volume of fluid, such asblood, from the patient 350.

Because the tube 12 may be filled substantially directly from thepatient 350, the collection of the fluid, such as blood, may be providedsubstantially efficiently to the tube 12. Although any appropriatemechanism may be used to assist in withdrawing the blood from thepatient 350 the vacuum system 356 may be provided including the vacuuminducing member 358. Any appropriate vacuum creating device may be used,such as a mechanical pump or the like. Nevertheless, the tube 12 may befilled for use during a selected procedure.

As discussed above, the tube 12 may be used to separate a selectedportion of the blood obtained from the patient 350 substantiallyintraoperatively. Therefore, the collection or separation of the variouscomponents may be substantially autologous and substantiallyintraoperatively. Moreover, obtaining the fluid directly from thepatient 350 may increase the efficiency of the procedure and theefficiency of the intraoperative or the operative procedure.

With reference to FIG. 9, the separator 10 may be used to separate anyappropriate material. The material may be separated for any purpose,such as a surgical procedure. For example, a selected fraction of a bonemarrow aspirate or a bone marrow portion may be produced with theseparator 10 according to various embodiments. The selected fraction ofthe bone marrow aspirate may include various components, such asundifferentiated cells. The various undifferentiated cells may bepositioned in a selected scaffold or relative to a selected portion of apatient for providing a volume of the undifferentiated cells to thepatient. It will be understood that the method described according toFIG. 9 is merely exemplary of various embodiments that may be used toprovide a selected fraction of a bone marrow aspirate or other materialto a patient or selected position. The selected portion can be placed onthe scaffold by a method, including spraying, painting, dipping, orcombinations thereof.

A method of selecting or creating a selected fraction of a bone marrowaspirate in a selected scaffold according to a method 400 is illustratedin FIG. 9. Generally, the method 400 may start in block 402 in obtaininga bone marrow aspirate volume. The bone marrow aspirate (BMA) may beobtained in any selected or generally known manner. For example, aselected region of bone, such as a portion near an operative procedure,may be used to obtain the bone marrow aspirate. Generally, an accessingdevice, such as a syringe and needle, may be used to access anintramedullary area of a selected bone. The BMA may then be withdrawninto the syringe for various procedures. Once a selected volume of theBMA is obtained in block 402, the BMA may be positioned in the separator10 according to various embodiments in block 404. The BMA may bepositioned in any appropriate separator, such as those described aboveincluding the separator 10. Once the BMA is positioned in the separator10, a selected fraction of the BMA may be separated from the BMA inblock 406.

The selected fraction of the BMA may include undifferentiated cells orany appropriate portion of the BMA. The fractionation or separation ofvarious fractions of the BMA may allow for a volume of BMA to be takenfrom a single location and the separation or concentration of theselected portion may be performed in the separator 10. Generally,obtaining a small volume of the selected portion from a plurality oflocations may be used to obtain an appropriate volume of BMA or selectedfraction of the BMA. Nevertheless, the separator 10 may allow forseparating a selected volume from a single location from which the BMAis obtained. This may reduce the time of a procedure and increase theefficiency of obtaining the selected fraction of the BMA.

In addition to obtaining a volume of the BMA in block 402, a volume ofwhole blood may be obtained in block 408. The volume of blood obtainedin block 408, according to any appropriate procedure, including thosedescribed above, may then be positioned in the separator 10, in block410. The whole blood may be positioned in any appropriate separator,such as those described above or a separator to separate a selectedfraction of the whole blood. As described above, the whole blood may beseparated into an appropriate fraction, such as a fraction including aplatelet portion or buffy coat. The whole blood may be separated intoselected fractions in block 412. It will be understood that the BMA andthe whole blood volume may be obtained substantially simultaneously orconsecutively in block 402 and 408. Similarly, the selected fractions ofthe BMA obtained in block 406 and whole blood obtained in block 412 mayalso be performed substantially sequentially or simultaneously. Forexample, the separator 10 including the volume of the BMA may bepositioned in a separating device, such as a centrifuge, substantiallyopposite, so as to balance, the separator 10 including the volume of thewhole blood. Therefore, a single separation, such as centrifugeprocedure may be used to separate both the BMA and the whole blood intoselected fractions. This again may increase the efficiency of theprocedure to provide both a selected fraction of the BMA and a selectedfraction of the whole blood substantially simultaneously.

The selected fractions of the BMA and the whole blood, provided in block406 and 412 may be harvested in block 414. The selected fractions of theBMA and the whole blood, may be harvested in block 414 for appropriatepurposes, such as those described herein. The separator 10 may be usedto obtain the selected fractions of the BMA and the whole blood, throughvarious procedures, such as those described above.

After harvesting the selected fractions of the BMA and the whole bloodin block 414, the selected fraction of the BMA may be positioned on anappropriate scaffold in block 416. The scaffold in block 416 may be anyappropriate scaffold. The undifferentiated cells of the BMA may allowfor a substantial source of cells for use during a substantially naturalhealing after an operative procedure, for example, the natural healingof a patient may use the supplied undifferentiated cells. Therefore, thescaffold may be positioned in a selected portion of the anatomy and thecells may be allowed to grow and differentiate into selected portions inthe implanted position.

In addition to positioning the selected fraction of the BMA and thescaffold in block 416, the platelets of the whole blood may bepositioned on or near the scaffold of block 418. The platelets of thewhole blood fraction positioned in the scaffold of block 418 may assistthe undifferentiated cells and the anatomy into which the scaffold ispositioned to allow for a substantially efficient and complete healing.The platelet fraction of the whole blood sample may include varioushealing and growth factors that may assist in providing an efficient andproper healing in the anatomy. Therefore, the undifferentiated cells ofthe BMA, or other selected fraction obtained from the separation of theBMA, and the selected fraction of the whole blood, obtained from theseparator, may be used with the scaffold to provide a substantiallyefficient implant. In addition, the separator 10, or any appropriateseparator, such as that described above, may allow for a substantiallyquick and efficient separation of the BMA and the whole blood into anappropriate fraction for use in the procedure.

After the selected portion of the BMA and the whole blood are positionedon the scaffold in blocks 416 and 418 the scaffold may be implanted inblock 420. As described above, the scaffold may be implanted in anyappropriate position in the block 420 for various procedures. It will beunderstood that the scaffold may be implanted for any appropriateprocedure and may allow for positioning the selected portion of the BMA,such as undifferentiated cells, and the selected portion of the wholeblood, such as platelets, relative to a selected portion of the anatomy.The scaffold may allow for a bone in growth, such as allowed with theundifferentiated cells, to assist in healing of a selected portion ofthe anatomy.

The description of the invention is merely exemplary in nature and,thus, variations that do not depart from the gist of the invention areintended to be within the scope of the invention. Such variations arenot to be regarded as a departure from the spirit and scope of theinvention.

1. A method of separating a multi-component fluid using a centrifugeprocess and buoy system, including a first and a second piston, in acontainer to hold the multi-component fluid during the centrifugeprocess, comprising: interconnecting the first piston and the secondpiston with a connection member; forming a first fraction and a secondfraction by centrifuging the multi-component fluid disposed in thecontainer; containing at least a portion of the second fraction in acollection area of the first piston; disposing the first piston relativeto the second piston; and moving the second piston relative to the firstpiston to at least assist in withdrawing the second fraction.
 2. Themethod of claim 1, further comprising withdrawing at least a portion ofthe second fraction including: forming a cannula in the connectionmember; and drawing the portion of the second fraction through thecannula.
 3. The method of claim 1, further comprising; moving the firstpiston and the second piston substantially as a system through themulti-component fluid; wherein a first fraction is disposed on a side ofat least one of the first piston and the second piston and the secondfraction is disposed substantially between the first piston and thesecond piston.
 4. The method of claim 1, wherein the collection area ofthe first piston includes a first face that is substantiallycomplementary to a second face of the second piston, such that when thesecond piston including the second face engages the first face of thefirst piston substantially no volume remains in the first face; whereinwithdrawing the second fraction includes moving the second face of thesecond piston into the first face of the first piston.
 5. The method ofclaim 1, wherein said first fraction and said second fraction are formedfrom at least one of a whole blood sample, a bone marrow aspirate, orcombinations thereof.
 6. The method of claim 1, further comprising:withdrawing a selected volume of a material including themulti-component fluid from a patient and positioning the volume of thematerial relative to the first piston and the second piston.
 7. Themethod of claim 6, further comprising: collecting the material from thepatient and placing it directly relative to the first piston and thesecond piston.
 8. The method of claim 7, further comprising: forming apressure relative to the first piston and the second piston lower than apressure of the material within the patient.
 9. A method of separating amulti-component fluid using a centrifuge process and buoy system,including a first and a second piston, in a container to hold themulti-component fluid during the centrifuge process, comprising:interconnecting the first piston and the second piston with a connectionmember; forming a first fraction and a second fraction by centrifugingthe multi-component fluid disposed in the container; containing at leasta portion of the second fraction in a collection area of the firstpiston; and disposing the first piston relative to the second piston;wherein interconnecting the first piston and the second piston with theconnection member further includes: fixing the connection member to thefirst piston; and slidably connecting the second piston to theconnection member; wherein the second piston is operable to moverelative to the first piston along the connection member.
 10. The methodof claim 9, further comprising: providing a buoy system within thecontainer; wherein said buoy system includes the first piston and thesecond piston operably interconnected with the connection member. 11.The method of claim 9, wherein containing the second fraction in thecollection area of the first piston includes: moving the first pistonthrough the multi-component fluid in conjunction with the second pistonto collect at least the second fraction in the collection area.
 12. Themethod of claim 9, further comprising: withdrawing at least a portion ofthe second fraction.
 13. A system for separating and extracting selectedfractions from a container, the system comprising: a piston system,including: a first piston having a first density; a second piston havinga second density; a connection member operably interconnecting saidfirst piston and said second piston; a first piston collection facedefined by said first piston; wherein a selected fraction is operable tobe collected in said first piston collection face between said firstpiston and said second piston; wherein said second piston defines asecond piston collection face; wherein said second piston collectionface is substantially complementary to said first piston collectionface, such that said second piston collection face may substantiallymate with said first piston collection face to substantially eliminateany volume defined between said second piston collection face and saidfirst piston collection face.
 14. The system of claim 13, furthercomprising: a container defining an internal diameter; wherein saidpiston system is disposable within said container during a centrifugeprocess.
 15. The system of claim 13, wherein said first pistoncollection face is a substantially concave surface defined by an upperportion of said first piston; wherein said first piston collection faceis disposed between said first piston and said second piston.
 16. Thesystem of claim 13, wherein said connection member further defines aport extending between said first piston collection face and saidcannula defined by said connection member.
 17. The system of claim 13,wherein said first piston collection face includes an angle of concavityof above 0.5° to about 45° relative to a central axis of said connectionmember.
 18. The system of claim 15, wherein said second piston includesan upper surface defining an angle relative to a central axis of saidconnection member of about 90° to about 150°.
 19. The system of claim13, wherein said second piston defines a second piston collection face;wherein of said second piston collection face is substantiallycomplementary to said first piston collection face.
 20. The system ofclaim 13, further comprising a container operable to contain said pistonsystem.
 21. The system of claim 20, further comprising a vacuum creatingsystem interconnected with said container to form a pressuredifferential in said container relative to a position exterior to saidcontainer.
 22. The system of claim 21, wherein the vacuum creatingsystem further comprises a resilient bulb interconnected with at least afirst valve such that said resilient bulb is operable to withdraw avolume of fluid from said container and expel the volume of fluid fromsaid container while substantially eliminating the re-entry of a volumeof fluid into said container.
 23. The system of claim 20, furthercomprising: a fluid accepting port formed in the container; and aconduit interconnecting said fluid accepting port and a fluid source.24. The system of claim 23, wherein said conduit is operable to beinterconnected with an intravenous system such that a material isoperable to be collected directly from an intravenous area into saidcontainer.
 25. The system of claim 13, wherein said second pistonincludes a center axis and an upper surface defining an angle of about70° to about 90° relative to said center axis.
 26. The system of claim25, wherein the container further comprises a top wall of the containerand a port extending from the top wall; and a flexible tube extendingfrom the port to a collection member.
 27. A system for separating andextracting selected fractions from a container, the system comprising: apiston system, including: a first piston having a first density; asecond piston having a second density; a connection member operablyinterconnecting said first piston and said second piston; a first pistoncollection face defined by said first piston; wherein a selectedfraction is operable to be collected in said collection face betweensaid first piston and said second piston; wherein said second piston ismovable relative to said first piston along said connection member. 28.The system of claim 27, wherein an extraction member is operablyconnected to and operable to produce a force through said cannula, suchthat said second piston is urged toward said first piston to assist inextraction of the second fraction.
 29. A system for separating andextracting a selected fraction from a container, the system comprising:a container having a container central axis; a buoy system, including: afirst buoy, a second buoy, and a connection member operable to bepositioned in the container; a first collection face defined by saidfirst buoy which includes an upper surface defining an angle relative toa buoy central axis defined as extending through the first buoy of about90° to about 150°; and a second collection face defined by the secondbuoy substantially complementary to the first collection face such thatsaid second collection face may substantially mate with said firstcollection face to substantially eliminate any volume defined betweensaid second collection face and said first collection face; wherein thefirst buoy has a first density; wherein the second buoy has a seconddensity; wherein the connection member connects the first buoy and thesecond buoy; wherein the container central axis and the buoy centralaxis are operable to be aligned; wherein a selected fraction is operableto be collected in the first collection face between the first buoy andthe second buoy; wherein the connection member defines a passage with aportal operable to gain access to the first collection face.
 30. Thesystem of claim 29, further comprising: a centrifuge device; wherein thecontainer and the buoy system are operable to be positioned in thecentrifuge device.
 31. The system of claim 30, wherein the first densityof the first buoy and the second density of the second buoy aredifferent such that the first buoy and the second buoy are operable tobe separated with a force formed with the centrifuge device.